Activation of defence mechanisms in higher eukaryotes is dependent on an array of pattern recognition receptors (PRRs) which recognize molecular structures that are characteristic for microbes. These pathogen-associated molecular patterns (PAMPs) play key roles as activators of the innate immune response in animals (Medzhitov and Janeway, 2002; Akira and Takeda, 2004) and, analogously, as elicitors of defence responses in plants (Nornberger et al., 2004). In contrast to animals, plants have no specialized cells or organs for defence; rather, all cells can mount a defence response upon pathogen perception. Perception of pathogens by plants can be divided into three main phases which appear to reflect steps of co-evolution in plant-pathogen interaction (Nürnberger et al., 2004). In a first phase, perception of PAMPs or “general elicitors” by the host leads to rapid activation of defence mechanisms such as cell wall reinforcement by callose deposition and production of reactive oxygen species (ROS), and induction of numerous defence-related genes. In a second phase, virulence factors evolved by successful pathogens can inhibit these PAMP-elicited basal defences (Espinosa and Alfano, 2004; Nomura et al., 2005; Kim et al., 2005). In a third phase, in turn, certain plant cultivars have evolved resistance (R) proteins specialized to detect these pathogen-derived virulence factors or the effects caused by them. As a consequence of this R protein-dependent perception process these plants trigger full, irreversible defence resulting in local lesion of the tissue termed hypersensitivity response (HR) and arrest of pathogen spreading (Nimchuk et al., 2003; Jones and Takemoto, 2004).
PAMPs perceived by plants include structures characteristic for oomycetes like the cell wall β-glucan, the pep13 epitope conserved in cell wall transglutaminase and secreted lipotransfer proteins termed elicitins (Nürnberger et al., 2004). Plants can also perceive structures signalling the presence of true fungi like the cell wall polysaccharide chitin and the fungal sterol ergosterol. Similarly, plants have been reported to recognize structures characteristic for bacteria like lipopolysacharides (LPS), bacterial cold-shock protein (CSP), flagellin and EF-Tu (Nürnberger et al., 2004; Zipfel and Felix, 2005). Some of these PAMPs are only perceived by a narrow range of plant species, whereas others trigger defence responses in a very broad range of species. Typically, any given plant seems to have perception systems for several PAMPs characteristic of the same class of microorganism. For example, flagellin induces responses in plants belonging to many different orders, while perception of the additional bacterial PAMPs CSP and EF-Tu seems to be restricted to the orders of Solanales and Brassicales, respectively.
Whereas many microbial patterns can act as PAMPs in plants, the corresponding PRRs remain largely unknown. So far, receptor binding sites have been reported only for a few examples, notably for heptaglucan from oomycetes (Umemoto et al., 1997), fungal xylanase (Ron and Avni, 2004) and bacterial flagellin (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2006). The Arabidopsis flagellin receptor FLS2 is a membrane bound receptor kinase with a leucine-rich repeat (LRR) domain facing the apoplast. As such, FLS2 is a member of the receptor like kinases (RLKs), a family comprising >600 members in Arabidopsis (Shiu et al., 2004). So far, only a few of these RLKs have been associated with specific functions but these examples clearly indicate that members of this large gene family function as receptors for many of the different extracellular signals perceived by plants (Dievart and Clark, 2003; Shiu et al., 2004). Similar to FLS2, some of the RLKs with unknown function might act as PRRs for other PAMPs such as EF-Tu.
The epitope of EF-Tu, which gets recognized by Arabidopsis is restricted to a small domain around the acetylated N-terminus of the mature protein. PAMP-activity of EF-Tu can be mimicked by synthetic peptides representing this N-terminus with a minimum of the first 18 amino acid residues, as in the peptides elf18 and elf26 (Kunze et al., 2004). These peptides are active at subnanomolar concentrations while shorter peptides comprising less than 18 residues exhibit lower activity, and elf12, a peptide with only 12 residues, is completely inactive as an inducer of responses. Interestingly, elf12 acts as a specific, competitive antagonist for EF-Tu, indicating that this peptide binds but does not activate the EF-Tu receptor (Kunze et al., 2004). All these characteristics of EF-Tu perception bear resemblance to the perception of flagellin by its receptor FLS2.
In a first aspect of this invention, we demonstrate that EF-Tu binds to specific, high-affinity receptor binding sites distinct from FLS2 but elicits a set of defence response that is highly similar to that induced by flagellin.
In a second aspect of this invention, we screened T-DNA insertion lines for various FLS2-related RLKs and identified EFR (EF-Tu Receptor) as required for perception of EF-Tu in Arabidopsis. Nicotiana benthamiana plants have no perception system for EF-Tu, but gained capacity to respond to this PAMP when expressing EFR.
In a third aspect of this invention, we demonstrate that mutants lacking EF-Tu perception are more susceptible to transformation by Agrobacterium tumefaciens, thus revealing the functional importance of this perception system for plant defence. Furthermore, this result suggests that differences in sensing PAMPs exposed by A. tumefaciens might explain the pronounced differences in susceptibility to transformation by this bacterium observed between different plant species.
In other aspects, it is demonstrated that inhibitors of callose deposition, such as xanthan gum and 2-deoxy-D-Glucose (2-DDG) can increase the efficiency of A. tumefaciens-mediated transient expression.
Other aspects of this invention will be apparent from a review of the full disclosure and the claims appended hereto.